Live cell imaging by multifocal multiphoton microscopy

Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.     

The first decade of using multiphoton microscopy for high-power kidney imaging

In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans.     

Simultaneous multiple-excitation multiphoton microscopy yields increased imaging sensitivity and specificity

Background  

Multiphoton microscopy (MPM) offers many advantages over conventional wide-field and confocal laser scanning microscopy (CLSM) for imaging biological samples such as 3D resolution of excitation, reduced phototoxicity, and deeper tissue imaging. However, adapting MPM for critical multi-color measurements presents a challenge because of the largely overlapping two-photon absorption (TPA) peaks of common biological fluorophores. Currently, most multi-color MPM relies on the absorbance at one intermediate wavelength of multiple dyes, which introduces problems such as decreased and unequal excitation efficiency across the set of dyes.     

New developments in multiphoton microscopy

Multiphoton laser-scanning microscopy is still developing rapidly, both technologically and by broadening its range of application. Technical progress has been made in the optimization of fluorophores, in increasing the imaging depth of multiphoton microscopy, and in microscope miniaturization. These advances further facilitate the study of neuronal structure and signaling in living and even in behaving animals, in particular in combination with the expression of fluorescent proteins. In addition, nonlinear optical contrast mechanisms other than multiphoton excitation of fluorescence are being explored.     

In vivo brain imaging of mitochondrial Ca2+ in neurodegenerative diseases with multiphoton microscopy

Mitochondria are involved in a large number of essential roles related to neuronal function. Ca²⁺ handling by mitochondria is critical for many of these functions, including energy production and cellular fate. Conversely, mitochondrial Ca²⁺ mishandling has been related to a variety of neurodegenerative diseases. Investigating mitochondrial Ca²⁺ dynamics is essential for advancing our understanding of the role of intracellular mitochondrial Ca²⁺ signals in physiology and pathology. Improved Ca²⁺ indicators, and the ability to target them to different cells and compartments, have emerged as useful tools for analysis of Ca²⁺ signals in living organisms. Combined with state-of-the-art techniques such as multiphoton microscopy, they allow for the study of mitochondrial Ca²⁺ dynamics in vivo in mouse models of the disease. Here, we provide an overview of the Ca²⁺ transporters/ion channels in mitochondrial membranes, and the involvement of mitochondrial Ca²⁺ in neurodegenerative diseases followed by a summary of the main tools available to evaluate mitochondrial Ca²⁺ dynamics in vivo using the aforementioned technique.     

When multiphoton microscopy sees near infrared

The need for quantification and real time visualization of developmental processes has called for increasingly sophisticated imaging techniques. Among them, multiphoton microscopy reveals itself to be an extremely versatile tool owing to its unique ability to combine fluorescent imaging, laser ablation, and higher harmonic generation. Furthermore, recent advances in femtosecond lasers and optical parametric oscillators (OPO) are now opening doors for imaging at unprecedented wavelengths centered in the tissue transparency window. This Review describes promising multiphoton approaches using OPO and the growing number of useful applications of non-linear microscopy in the field of developmental biology. Basic characteristics associated with these techniques are described along with the main experimental challenges when applied to embryo imaging.     

Immunoelectron microscopy in embryos

Immunogold labeling of proteins in sections of embryos embedded in acrylate media provides an important analytical tool when the resolving power of the electron microscope is required to define sites of protein function. The protocol presented here was established to analyze the role and dynamics of the activated protein kinase C/Rack1 regulatory system in the patterning and outgrowth of limb bud mesenchyme. With minor changes, especially in the composition of the fixative solution, the protocol should be easily adaptable for the postembedding immunogold labeling of any other antigen in tissues of embryos of diverse species. Quantification of the labeling can be achieved by using electron microscope systems capable of supporting digital image analysis.     

Functional studies in living animals using multiphoton microscopy

In vivo microscopy is a powerful method for studying fundamental issues of physiology and pathophysiology. The recent development of multiphoton fluorescence microscopy has extended the reach of in vivo microscopy, supporting high-resolution imaging deep into the tissues and organs of living animals. As compared with other in vivo imaging techniques, multiphoton microscopy is uniquely capable of providing a window into cellular and subcellular processes in the context of the intact, functioning animal. In addition, the ability to collect multiple colors of fluorescence from the same sample makes in vivo microscopy uniquely capable of characterizing up to three parameters from the same volume, supporting powerful correlative analyses. Since its invention in 1990, multiphoton microscopy has been increasingly applied to numerous areas of medical investigation, providing invaluable insights into cell physiology and pathology. However, researchers have only begun to realize the true potential of this powerful technology as it has proliferated beyond the laboratories of a relatively few pioneers. In this article we present an overview of the advantages and limitations of multiphoton microscopy as applied to in vivo imaging. We also review specific examples of the application of in vivo multiphoton microscopy to studies of physiology and pathology in a variety of organs including the brain, skin, skeletal muscle, tumors, immune cells, and visceral organs.     

Nonlinear magic: multiphoton microscopy in the biosciences

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.     

Picosecond multiphoton scanning near-field optical microscopy.

We have implemented simultaneous picosecond pulsed two- and three-photon excitation of near-UV and visible absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 1064-nm emission from a pulsed Nd:YVO4 laser was used to excite the visible mitochondrial specific dye MitoTracker Orange CM-H2TMRos or a Cy3-labeled antibody by two-photon excitation, and the UV absorbing DNA dyes DAPI and the bisbenzimidazole BBI-342 by three-photon excitation, in a shared aperture SNOM using uncoated fiber tips. Both organelles in human breast adenocarcinoma cells (MCF 7) and specific protein bands on polytene chromosomes of Drosophila melanogaster doubly labeled with a UV and visible dye were readily imaged without photodamage to the specimens. The fluorescence intensities showed the expected nonlinear dependence on the excitation power over the range of 5-40 mW. An analysis of the dependence of fluorescence intensity on the tip-sample displacement normal to the sample surface revealed a higher-order function for the two-photon excitation compared to the one-photon mode. In addition, the sample photobleaching patterns corresponding to one- and two-photon modes revealed a greater lateral confinement of the excitation in the two-photon case. Thus, as in optical microscopy, two-photon excitation in SNOM is confined to a smaller volume.     

Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.     

Label-free imaging of lipid-droplet intracellular motion in early Drosophila embryos using femtosecond-stimulated Raman loss microscopy

Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed "femtosecond-stimulated Raman loss" microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.     

Spectral and lifetime fluorescence imaging microscopies: new modalities of multiphoton microscopy applied to tissue or cell engineering

Spectral and multiphoton imaging is the preferred approach for non-invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross-talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM-FRET).     

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.     

Digital microscopy for multiparameter FISH imaging

Fluorescence in situ hybridization (FISH) allows the direct localization of DANN and RNA sequences on chromosomes, in cells and in tissue. The technique is based on hybridization between target sequences of single-strand DNA of chromosomes or cell nuclei with marked complementary specimens. The signal is amplified using fluorochrome-marked specific antibodies and made visible under a microscope. Signals from painted chromosomes, stained subchromosomal regions or localized single probes are generally visible when an epifluorescence microsope is used. In order to view and display different fluorochromes, single filter sets, as well as double and triple bandpass filters, are in use. For multiple fluoroscence imaging, lenses with high numerical aperture, mostly oil immersion systems, are recommended. In conventional photomicrography, triple exposure on high-speed film (e.g., 400-1,000 ASA) is more or less the limitation. Opto=electronic methods using a CCD and laser summing techniques have considerably extended the application range of multiple fluorescence techniques. By means of digitized images, simultaneous detection of multiple-labelled objects and ratio imaging up to 24 colors are possible today. Current FISH approaches are based on chromosome-painting probes to distinguish all 24 chromosomes by their unique spectral signatures.     

Culturing of avian embryos for time-lapse imaging

Monitoring morphogenetic processes, at high resolution over time, has been a long-standing goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however, imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h.     

A compact multiphoton 3D imaging system for recording fast neuronal activity

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license.     

Intraocular multiphoton microscopy with subcellular spatial resolution by infrared femtosecond lasers

We reported on the in situ nonlinear optical sectioning of the corneal and retinal tissues based on the multiphoton microscopy (MPM) with different excitation wavelengths of infrared femtosecond (fs) lasers. The multiphoton nonlinear processing including two-photon fluorescence (2PF) and second harmonic generation (SHG) was induced under condition of high light intensities on an order of MW-GW/cm². The laser beams emitted from the solid-state Ti: sapphire systems were focused in a 0.1 femtoliter focus volume of a high numerous aperture diffraction-limited objective (40 × 1.3 N.A., oil). The corneal layers have been visualized using nonlinear optical tomography. In particular, corneal Bowman’s layer was optically determined in situ. The cellular and collagen components of tissues were selectively displayed with submicron spatial resolution and high efficiency without any assistance of staining or slicing. The preliminary study on retinal optical tomography is here also reported. MPM is a promising and convenient non-invasive technique by which the tissue layers can be visualized and the selective displaying of the tissue microstructures be realized. The optical biopsy based on intrinsic emission of MPM yields details that provide three-dimensional displaying of the tissue component and even have the potential to be used in clinical diagnostics.Dedicated on the occasion of the 66th birthday of Professor Dr. Karl-Juergen Halbhuber